Modified vaccinia Ankara expressing p53 in cancer immunotherapy

ABSTRACT

Mutations to the tumor suppressor protein p53 have been observed in 40-60% of all human cancers. These mutations are often associated with high nuclear and cytoplasmic concentrations of p53. Since many tumors exhibit highly elevated p53 levels, the protein is an attractive target for cancer immunotherapy. Unfortunately, p53 is an autoantigen that is likely to be tolerated as a self-protein by the immune system. The present invention is based on the discovery that this self-tolerance can be overcome by administration of recombinant modified vaccinia Ankara (MVA) containing a nucleic acid that encodes p53 (rMVAp53). The invention discloses a method of generating a p53-specific CTL-response to tumor cells expressing mutated p53 by administering a composition comprising rMVAp53. Administration of rMVAp53 decreases tumor development, tumor growth, and mortality in a variety of malignant cell types. These effects are enhanced by administration of CTLA-4 blocker and/or CpG oligodeoxynucleotide immunomodulators.

BACKGROUND OF INVENTION

The present utility application claims priority to provisional patentapplication U.S. Ser. No. 60/436,268 (Ellenhorn and Diamond), filed Dec.23, 2002, and U.S. Ser. No. 60/466,607 (Ellenhorn and Diamond), filedApr. 30, 2003, the disclosures of which are incorporated by reference intheir entirety herein.

GOVERNMENT INTEREST

This invention was made with government support in part by grants fromthe NIH, Division of AIDS (RO1-Al43267 and R21-Al44313) and NCI:RO1:CA77544, PO1-CA30206, R29-CA70819, and CA33572. The government mayhave certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the fields of virology, molecularbiology, and tumor immunology. Specifically, this invention relates tocompositions and methods for eliciting immune responses effectiveagainst malignancies expressing p53.

BACKGROUND

p53 is a tumor suppressor protein that regulates the expression ofcertain genes required for cell cycle arrest or apoptosis. The tumorsuppressor gene encoding p53 is activated by DNA damage, cell stress, orthe aberrant expression of certain oncogenes (Levine 1997). Onceactivated, wild type p53 (wt p53) serves to temporarily arrest the cellcycle, allowing time for DNA repair and preventing cells with damagedDNA from proliferating uncontrollably (Levine 1997). p53 is alsoinvolved in inducing apoptosis in cells with certain types ofphysiologic damage (Levine 1997).

Mutations in p53 that functionally inactivate its growth suppressingability have been observed in 40-60% of all human cancers, and areassociated with the malignant phenotype (Hainaut 2000). Mutations to p53occur as early events in tumorigenesis (Millikan 1995; Querzoli 1998;Allred 1993), abrogating the ability of the protein to suppress celldivision (Finlay 1989; Eliyahu 1989). The regulation of p53 expressionin cells can occur at the level of p53 mRNA abundance or at the level ofp53 protein abundance. Mutations of p53 are often associated with highnuclear and cytoplasmic concentrations of the p53 protein, due to theprolonged half-life of the mutated protein. Many tumors arecharacterized by highly elevated intracellular p53 levels compared tononmalignant cells. Other tumors synthesize large amount of mutated p53,but contain low or below normal steady-state levels of intracellularp53, presumably as a result of accelerated intracellular degradation ofthe protein. Overexpression of p53 is an independent predictor of moreaggressive cancers (Turner 2000; Elkhuizen 2000; Zellars 2000), lymphnode metastases (Pratap 1998), failure to respond to standard therapies(Berns 1998; Berns 2000), and mortality (Sirvent 2001; Querzoli 2001).

Missense point mutations are the most frequent p53 mutations in cancer,leaving the majority of the p53 protein in its wild type form (wt p53).Although p53 mutations may represent true tumor specific antigens, mostof these mutations occur at sites that do not correspond to immunologicepitopes recognized by T cells (Wiedenfeld 1994). Because of this, anywidely applicable p53-directed immunotherapy must target wt p53. Inexperimental models, it has been possible to target p53 because themutated molecule is associated with high nuclear and cytoplasmicconcentrations of the p53 protein (Finlay 1988). p53 is an attractivetarget for adaptive immune response because the intracellularconcentration of nonmutated p53 in healthy cells is very low (Zambetti1993; Reich 1984). This means that healthy cells expressing non-mutantp53 will most likely escape an enhanced immune response toover-expressed mutant p53 (Offringa 2000).

p53, like most tumor associated antigens that are recognizable by thecellular arm of the immune system, is an autoantigen (Rosenberg 2001).The fact that p53 is an autoantigen widely expressed throughoutdevelopment (Schmid 1991), coupled with the fact that the majority ofmutated p53 being expressed in tumors has the same structure as the wildtype protein, means that tumor-expressed p53 is likely to be toleratedas a self-protein by the immune system. This tolerance, which has beenshown by functional and tetramer studies in mice to exist at thecytotoxic T lymphocyte level (CTL) (Theobald 1997; Erdile 2000), limitsthe effectiveness of p53-directed immunotherapies. To be successful, aneffective immunotherapy must overcome this tolerance without alsoinducing autoimmunity against normal cells and tissues (Theobald 1997;Erdile 2000; Hernandez 2000). Small numbers of self-reacting T cellsescape during the processes involved in the immune tolerance.

Tumors overexpressing p53 have been eliminated in murine models by thesystemic administration of epitope specific CTL (Vierboom 2000a;Vierboom 2000b; Vierboom 1997; Hilburger 2001), epitope pulsed dendriticcells (DC) (Mayordomo 1996), or mutant p53 epitope with IL-12(Noguchi-1995). Each of these strategies has considerable drawbacks withregards to clinical applicability. CTL infusion and infusion of epitopepulsed dendritic cells are time consuming and expensive, because theisolation, culturing, and reinfusion of cells must be performed individually for each patient. Conversely, in order to produce any effect,the cell-free vaccination strategies previously used required eitherintratumoral injections or vaccination prior to tumor challenge, neitherof which represents a practical approach in the clinical setting. Thereis thus a need for simplified, efficient, and widely applicableimmunotherapeutic strategies in the treatment of cancer.

SUMMARY OF THE INVENTION

The p53 gene product is overexpressed in a majority of cancers, makingit an ideal target for cancer immunotherapy. The efficacy of thesetherapies has been limited, however, by the fact that tumor-expressedp53 is likely to be tolerated as a self-protein by the immune system.The present invention is based on the discovery that this self-tolerancecan be overcome by administration of recombinant MVA containing anucleic acid that encodes p53 (rMVAp53). Administration of p53 is shownto greatly decrease tumor development, tumor growth, and mortality inmice challenged with a variety of malignant cell types. It is also shownthat the therapeutic effects of rMVAp53 are enhanced by administrationof a CTLA-4 blocker or CpG oligodeoxynucleotide (CpG ODN)immunomodulator. This enhancement is greatest when both immunomodulatorsare administered. The present invention provides a recombinant MVAcomposition for use in the treatment of cancer, a method of treatingcancer using this composition, and a kit for administration of thecomposition.

In a first aspect, the present invention provides a compositioncomprising recombinant MVA that contains a nucleic acid encoding p53.Preferably, the p53 encoded by the recombinant MVA is wt human p53.According to the present invention, the composition may also contain aCTLA-4 blocker and/or a CpG ODN.

In another aspect, the present invention provides a method for treatinga subject having a p53-expressing malignancy. This method is based onadministration of a recombinant MVA containing a nucleic acid thatencodes p53. Preferably, the method also calls for administration of aCTLA-4 blocker and/or CpG ODN as an immunomodulator. In a third aspect,the present invention provides a kit for treating a p53-expressingmalignancy. This kit contains a recombinant MVA containing a nucleicacid that encodes p53, and may also contain a CTLA-4 blocker and/or CpGODN as an immunomodulator. In a final aspect, this invention providesfor an MVA recombination plasmid containing a nucleic acid insert thatencodes wt human p53.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: PCR analysis of the pLW22-hup53 construct. rMVAhup53 injected(lanes 1, 2) and wtMVA infected (lanes 3, 4) BHK cells were subjected tototal DNA extraction and PCR amplification using wtMVA (lanes 2, 4) orhup53 (lanes 1, 3) specific primers. The rMVAhup53 product was shown tohave no contaminating wtMVA.

FIG. 2: Expression of mup53 by cells infected with rMVAmup53. Cellsinfected with rMVAmup53 express mup53 at high levels, confirming thatMVA is a suitable vaccine vector. Cell lysates were subjected toSDS-PAGE and Western blotting. The lanes are designated as follows: 1)Meth A, unmanipulated. Meth A sarcoma cells, 2) HCMV IE1 exon4-rMVAinfected BHK cells, 3-4) rMVAmup53 infected BHK cells (loaded 0.125 ul,0.25 ul cell lysates respectively), 5) rAdp53, and 6) rAdpp65 infectedHEK 293 cells. All lanes were loaded with 20 μl sample unless indicatedspecifically.

FIG. 3: Generation of a p53-specific CTL response by rMVAmup53 in vitro.A single intraperitoneal (i.p.) vaccination with rMVAmup53 generates p53specific CTL responses that efficiently kill cells overexpressing p53.(a) Splenocytes from mice treated with rMVAmup53 were harvested at 14days and restimulated in vitro for 6 days with rAdp53 infected syngeneicLPS blasts. CTL activity was evaluated in a standard 4-h ⁵¹Cr releaseassay using rVVp53 (solid line) or rWpp65 (dashed line) infected 10.1cells. (b) Splenocytes from rMVAmup53 (solid line) or rMVApp65 (dashedline) vaccinated mice were harvested at 14 days following vaccinationand restimulated in vitro for 6 days with rAdp53 infected syngeneic LPSblasts. Cytotoxicity was measured against rVVp53 infected 10.1 cells.(c) Splenocytes harvested 14 days after rMVAmup53 (solid line) orrMVApp65 (dashed line) vaccination were restimulated in vitro for 6 daysusing syngeneic LPS blasts infected with rMVAp53. Cytotoxicity wasmeasured against Meth A cells by a standard 4-h ⁵¹Cr release assay.

FIG. 4: Effect of vaccination with rMVAmup53 on Meth A tumor prevention.Balb/c mice were injected subcutaneously (s.c.) with 5×10⁵ Meth A cells.On day 5, mice were vaccinated with either 5×10⁷ pfu of rMVAmup53(MVAp53) (n=16), 5×10⁷ pfu rMVApp65 (MVApp65) (n=16), or PBS (n=12). Thesurvival plot shows the proportion of surviving animals in each group asa function of days post tumor challenge. The improvement of the micevaccinated with rMVAmup53 over both control groups is statisticallysignificant (P<1) as determined by the log rank test.

FIG. 5: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAb onestablished Meth A tumors. Mice were injected s.c. with a rapidly lethaldose of 10⁶ Meth A cells. On days 6, 9, and 12 mice were injected i.p.with either anti-CTLA-4 mAb (CTLA4 mAb) or control mAb. On day 7, micewere vaccinated with either 5×10⁷ pfu rMVAp53 (MVAp53) or 5×1 pfurMVApp65 (MVAapp65). The survival plot shows the proportion of survivinganimals in each group. The survival advantage of mice vaccinated withrMVAp53 plus anti-CTLA-4 mAb (n=14) over control animals receivingrMVApp65 plus CTLA-4 (n=14), rMVAp53 plus control ab (n=14), or rMVApp65plus control ab (n=6) is statistically significant (P<0.001) asdetermined by the log rank test.

FIG. 6: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAb onestablished 11A-1 tumors. Balb/c mice were injected s.c. with 2×10⁶11A-1 cells (p=0.00044, comparing rMVAmup53 plus anti-CTLA-4 mAb to allother groups). Anti-CTLA-4 mAb 9H10 (CTLA4 mAb) or the control hamsterisotype matched polyclonal antibody (isotype matched Ab) were injectedi.p. on days 4, 7, and 10 at 100, 50, and 50 μg dose, respectively. Onday 5, mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu rMVApp65 (MVApp65), or PBS. Each line represents themean and standard deviation of eight mice.

FIG. 7: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAb onestablished MC-38 tumors. C57BL/6 mice were injected s.c. with 1×10⁶MC-38 cells (p=0.0001, comparing rMVAmup53 plus anti-CTLA-4 mAb to allother groups). Anti-CTLA-4 mAb 9H10 (CTLA4 mAb) or the control hamsterisotype matched polyclonal antibody (isotype matched Ab) were injectedi.p. on days. 4, 7, and 10 at 100, 50, and 50 μg dose, respectively. Onday 5, mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu rMVApp65 (MVApp65), or PBS. Each line represents themean and standard deviation of eight mice.

FIG. 8: Effect of vaccination with rMVAmup53 plus CpG ODN on established11A-1 tumors. Balb/c mice were injected s.c. with 2×10⁶ 11A-1 cells(p=0.00002, comparing rMVAmup53 plus CpG ODN to all other groups). 15nmoles of CpG ODN (CpG) was injected i.p. on days 4, 9, and 14. On day5, the mice were immunized i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu of rMVApp65 (MVApp65), or PBS.

FIG. 9: Effect of vaccination with rMVAmup53 plus CpG ODN on establishedMeth A tumors. Balb/c mice were injected s.c. with 1×10⁶ Meth A cells(p=0.0015, comparing rMVAmup53 plus CpG ODN to all other groups). 15nmoles of CpG ODN (CpG) was injected i.p. on days 4, 9, and 14. On day5, the mice were immunized i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu of rMVApp65 (MVApp65), or PBS.

FIG. 10: Effect of vaccination with rMVAmup53 plus CpG ODN onestablished MC-38 tumors. C57BL/6 mice were injected with 1×10⁶ MC-38cells (p=0.0004, comparing rMVAmup53 plus CpG ODN to all other groups).15 nmoles of CpG ODN (CpG) was injected i.p. on days 4, 9, and 14. Onday 5, the mice were immunized i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu of rMVApp65 (MVApp65), or PBS.

FIG. 11: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAb andCpG ODN on established 11A-1 tumors. Balb/c mice (n=8) were injecteds.c. with 2×10⁶11A-1 cells. Anti-CTLA-4 mAb (CTLA4 mAb) was injectedi.p. on days 14, 17, and 20 at 100, 50, and 50 μg dose, respectively. 15nmoles of CpG ODN (CpG) was injected i.p. on days 14, 19, and 24. On day15, mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu rMVApp65, or PBS. The survival plot shows theproportion of surviving animals in each group as a function of days posttumor challenge. p=0.02 comparing combined CpG ODN and anti-CTLA-4 mAbto CpG ODN alone, and p=0.01 comparing combined CpG ODN and anti-CTLA-4mAb to anti-CTLA-4 mAb alone.

FIG. 12: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAb andCpG ODN on established MC-38 tumors. C57BL/6 mice (n=8) were injecteds.c. with 1×10⁶ MC-38 cells. Anti-CTLA-4 mAb was injected i.p. on days4, 7, and 10 at 100, 50, and 50 μg dose, respectively. 15 nmoles of CpGODN was injected i.p. on days 4, 9, and 14. On day 5, mice werevaccinated i.p. with either 5×10⁷ pfu rMVAmup53, 5×10⁷ pfu MVApp65, orPBS. The survival plot shows the proportion of surviving animals in eachgroup as a function of days post tumor challenge. p=0.002 comparingcombined CpG ODN and anti-CTLA-4 mAb to CpG alone, and p=0.001 comparingcombined CpG ODN and anti-CTLA-4 mAb with anti-CTLA-4 mAb alone.

FIG. 13: Cellular requirements for anti-CTLA-4 mAb immunomodulatoreffect on Meth A tumors. Balb/c mice (a) or IFN-γ^(KO) Balb/c mice (b)were injected s.c. with a rapidly lethal dose of 10⁶ Meth A cells.Groups of mice from both populations were injected i.p. with depletingdoses of anti-CD4, anti-CD8, anti-NK1.1, or control mAb on days —1, 1,3, and 10, and weekly thereafter. On days 6, 9, and 12 mice wereinjected i.p. with either anti-CTLA-4 mAb (CTLA4 mAb) or control mAb. Onday 7, mice were vaccinated with either 5×10⁷ pfu rMVAp53 (MVAp53) or5×10⁷ pfu rMVApp65 (MVAapp65). (a) Mean tumor growth was calculated foreach group of Balb/c mice, with error bars illustrating standarddeviation. The last datapoint for each line represents the firstmortality. (b) The proportion of surviving IFN-γ^(KO) Balb/c mice isplotted.

FIG. 14: Cellular requirements for CpG ODN immunomodulator effects on11A-1 tumors. Balb/c mice were injected s.c. with 2×10⁶ 11A-1 cells. 15nmoles of CpG ODN was injected i.p. on days 4, 9, and 14. On day 5, micewere vaccinated i.p. with 5×10⁷ pfu of rMVAmup53. Mice were injectedi.p. with depleting doses of anti-CD4 (CD4), anti-CD8 (CD8), anti-NK1.1(NK), or control mAb on days 4, 6, 8, and 15, and every 7 daysthereafter. Tumors were measured twice weekly in three dimensions.p=0.004 by two-sided Wilcoxon test, comparing CD8⁺ depleted to all othergroups. p=0.007, comparing anti-NK1.1 to anti-CD4 and control mAb.

FIG. 15: Cellular requirements for anti-CTLA-4 mAb immunomodulatoreffects on 11A-1 tumors. Mice were injected s.c. with 2×10⁶ 11A-1 cells.Anti-CTLA-4 mAb was injected i.p. on days 4, 7, and 10 at 100, 50, and50 μg/dose, respectively. On day 5, the mice were vaccinated i.p. with5×10⁷ pfu rMVAmup53. The mice were depleted of CD84, CD44, or NK cellsby i.p. injection with the relevant mAb or control mAb on days 4, 6, 8,and 15, and then every 7 days thereafter. Tumors were measured twiceweekly in three dimensions with calipers. Each curve represents the meanand standard deviation of 8 mice. p=0.004, comparing CD8⁺ depleted toall other groups. p=0.008, comparing CD4⁺ depleted to NK depleted andcontrol groups.

FIG. 16: Contribution of TLR 9 to the CpG ODN immunomodulator effect.TLR9^(−/−) (p=0.0009, comparing anti-CTLA-4 mAb to CpG ODN group) mutantC57BL/6 mice were injected s.c. with 1×10⁶ MC-38 cells. Mice weretreated with anti-CTLA-4 mAb (CTLA4 mAb) on days 4, 7, and 10 at 100,50, and 50 μg/dose, respectively, or with 15 nmoles of CpG ODN on days4, 9, and 14. On day 5, all mice were vaccinated i.p. with 5×10⁷ pfu ofrMVAmup53. Tumors were measured twice weekly in three dimensions withcalipers. Each curve represents the mean and standard deviation of 8mice.

FIG. 17: Contribution of IL-6 to the CpG ODN immunomodulator effect.IL-6^(−/−) (p=0.02, comparing anti-CTLA-4 mAb to CpG ODN group byWilcoxon 2-sided RankSum Test) mutant C57BL/6 mice were injected s.c.with 1×10⁶ MC-38 cells. Mice were treated with anti-CTLA-4 mAb (CTLA4mAb) on days 4, 7, and 10 at 100, 50, and 50 μg/dose, respectively, orwith 15 nmoles of CpG ODN on days 4, 9, and 14. On day 5, all mice werevaccinated i.p. with 5×10⁷ pfu of rMVAmup53. Tumors were measured twiceweekly in three dimensions with calipers. Each curve represents the meanand standard deviation of 8 mice.

FIG. 18: Expression of hup53 by cells infected with rMVAhup53. BHK cellswere injected with purified rMVAhup53 (MVA/p53). Expression of hup53 wasmeasured at 24 and 48 hours. Cell lysates were subjected to SDS-PAGE andWestern blotting. Lane 1: BHK cells injected with control MVA; Lane 2:BHK cells infected with rMVAhup53 for 24 hours; Lane 3: BHK cellsinfected with rMVAhup53 for 48 hours. All lanes were loaded with 20 μlof sample.

FIG. 19: Effect of vaccination with rMVAhup53 plus anti-CTLA-4 mAb andCpG ODN on established 4T1/hup53 tumors. Mice were injected s.c. with5×10⁴ 4T1/hup53, then vaccinated i.p. with 10⁷ pfu rMVAhup53 or PBScontrol on day 6. On day 16, mice received an rMVAhup53 or PBS boosterinjection, along with 1-5 nmole of CpG ODN and 50 μg of anti-CTLA-4 mAb.rMVAhup53 vaccinated mice displayed a significant improvement insurvival (p<0.05, two sided T-test) compared to PBS controls.

DETAILED DESCRIPTION

The present invention is based on the discovery that self-tolerance to aprotein expressed in both normal and cancerous cells can be overcome,and that a strong anti-tumor immune response can be generated withoutthe requirement for intratumoral administration and without theproduction of systemic toxicity or auto-immunity. The invention providesnovel cell-free compositions and methods for the generation of effectiveimmune responses against a wide variety of human malignancies,independent of the subject's haplotype or genotype. The examplesdiscussed below demonstrate that vaccination with a modified vacciniaAnkara vector engineered to express either wild type murine or wild typehuman p53 (rMVAmup53 or rMVAhup53) stimulates a vigorous p53-specificCTL response. This response can be enhanced by the co-administration ofan immunomodulator consisting of a CTLA-4 blocker and/or CpG ODN.

MVA virus (GenBank Accession Number U94848) is a variant of the Ankarastrain of vaccinia virus that was derived by over 570 serial passages onprimary chicken embryo fibroblast. Several properties of MVA as anattenuated poxvirus make it ideal for the generation of a therapeuticresponse to tumors expressing p53. One advantage of MVA is that it isable to efficiently replicate its DNA in mammalian cells, yet it isavirulent and does not propagate. This trait is the result of losing twoimportant host range genes among at least 25 additional mutations anddeletions that occurred during its passages through chicken embryofibroblasts (Meyer 1991; Antoine 1998). In contrast to NYVAC (attenuatedCopenhagen strain) and ALVAC (host range restricted avipox), both-earlyand late transcription in MVA are unimpaired, allowing for continuousgene expression throughout the viral life cycle (Carroll 1997a; Carroll1997b; Blanchard 1998; Sutter 1992). MVA has been found to be moreimmunogenic than the Western Reserve (WR) strain, and can be used inconditions of pre-existing poxvirus immunity (Ramirez 2000a; Ramirez2000b). The favorable clinical profile of MVA as a recombinant vaccinedelivery vehicle is buttressed by its benign safety profile as asmallpox vaccine in Europe in the late 1970's (Mayr 1999; Mayr 1978).MVA was administered to over 120,000 high-risk individuals, includingthe aged and very young, without serious side effects (Mayr 1978). Morerecently, MVA has also been administered to immunocompromised non-humanprimates without adverse outcome (Stittelaar 2001). This is in starkcontrast to other vectors, such as retroviruses and adenoviruses, whichpose documented risks to the human host. Immunotoxicity of the vector,adjuvant, or immunomodulator used is a particular point of concern inthe immunotherapy of cancer, as most cancer patients are severelyimmunocompromised due to chemotherapy, radiation, or theimmunosuppressive effects of the cancer itself. MVA was first developedinto a vaccine vehicle in the early 1990's, after it became clear thatnon-attenuated poxviruses such as the WR strain could not be safelyadministered to immunocompromised individuals (Redfield 1987; Collier1991). In summary, the potency of MVA as an expression vector combinedwith its safety profile in primates and humans make it highly attractiveas a delivery system for cancer genes.

Construction of rMVAmup53 and rMVAhup53 is achieved by recombinant DNAtechniques that are well known in the art (Sambrook et al., MolecularCloning, Cold Spring Harbor Laboratory, 2001; Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, 1986 and 2000). Thecoding sequence of wild type p53 can be conveniently obtained by RT-PCRusing p53-specific primers. These primers hybridize to DNA and serve asinitiation sites for DNA synthesis. Nucleotide primers are designed tobind at separate sites on opposing duplex strains, thereby defining theintervening sequence as the portion to be amplified. Nucleic acidmolecules to be employed as primers will generally include at least a 10base pair sequence complementary to the DNA segment to be amplified.Primer selection is well known to those of skill in the art. Primers forthe amplification of wt mup53 or wt hup53 can be designed to containappropriate restriction sites for subcloning into a suitable MVArecombination plasmid, such as pMCO3, pLW22, pLW51, PUCII LZ or otherMVA transfer vectors well known in the art. The recombination plasmidcontains sequences necessary for expression of the foreign gene insert,as well as the flanking sequences necessary for homologous recombinationinto a chosen-site of deletion in the MVA genome. To generaterecombinant MVA virus, cells are infected with MVA virus and transfectedwith the recombination plasmid containing the foreign gene insert. Afterhomologous recombination between virus and plasmid is allowed to occur,recombinant MVA expressing the inserted gene is isolated.

Cellular expression of p53 protein following infection with rMVAmup53 orrMVAhup53 was analyzed to determine the fidelity and extent of itsexpression from recombinant virus. Meth A cells, which overexpressmutated p53, were used as a positive control, and HCMV IE1 exon4 rMVAinfected BHK cells were used as a negative control. Western blotanalysis revealed abundant p53 expression by cells infected withrMVAmup53 or rMVAhup53, as well as by Meth A cells. No detectableexpression of p53 by HCMV IE1 exon 4-rMVA infected BHK cells wasobserved. High levels of p53 expression by rMVAp53 infected BHK cellswas also observed by fluorescence microscopy. The high level of p53expression exhibited by rMVAmup53 and rMVAhup53 compared to other viraland cellular forms demonstrates its usefulness in vaccination protocols.

In animal experimental models, MVA based vaccines stimulate tumorspecific CTL activity (Espenschied 2003; Drexler 1999) and effectregression of established tumors (Espenschied 2003; Carroll 1997b;Mulryan 2002; Rosales 2000). There are numerous advantages toimmunization with whole protein expressed in MVA. In contrast to peptideimmunization, multiple epitopes can be expressed, and a polyconal hostresponse can be stimulated. Antigen-specific cognate help, which isessential to the propagation of a CTL response, can be achieved throughexpression of a protein in MVA. In addition, expression of whole proteincan result in the stimulation of responses to otherwise crypticepitopes. Immunization with recombinant viruses may also avoid the needfor a complex and expensive approach involving the expansion andadoptive transfer of antigen-specific cells, or the need to generate anindividualized vaccine for a particular cancer patient. This advantageof a recombinant vaccine approach may encourage more widespread clinicaluse to prevent recurrence in patients with earlier stages of disease.

In vitro experiments were run to determine whether vaccination withrMVAmup53 could break p53 tolerance, resulting in the generation ofp53-specific CTL. Splenocytes were harvested from mice following asingle intraperitoneal (i.p.) vaccination with rMVAmup53, andrestimulated in vitro with p53 over-expressing cells. The splenocytesrecognized and lysed wt p53 over-expressing targets. In contrast,splenocytes from mice vaccinated with rMVApp65, which stimmulatesvigorous pp65 specific CTL responses, did not recognize the p53over-expressing targets, demonstrating the specificity of the lymphocyteresponse. rMVAmup53 vaccination can also stimulate CTL recognition ofMeth A cells, which express mutated p53. Restimulated splenocytes frommice vaccinated with rMVAmup53 recognized mutant p53 over-expressingMeth A, whereas control mice vaccinated with rMVApp65 did not.

Since a single vaccination with rMVAmup53 resulted in enhanced CTLresponse, there was sufficient justification to examine the effect ofrMVAmup53 vaccination on the growth of Meth A tumor cells in vivo.Administration of rMVAmup53 was shown to inhibit the outgrowth of murinesarcoma Meth A, an immunogenic tumor cells line that overexpressesmutant p53. Mice inoculated with a lethal dose of Meth A tumor cells andvaccinated with rMVAmup53 by i.p. injection three days later exhibitedslower tumor growth and higher survival rates than control animals. Amajority of the vaccinated mice failed to develop tumors entirely, andthese mice were resistant to rechallenge with Meth A after 52 days(Espenschied 2003).

The above results demonstrate the efficacy of a novel rMVAmup53cell-free vaccine at targeting p53 expressed by a malignant tumor.Additional experiments were performed to determine whether this effectcould be enhanced by addition of a CTLA-4 blocker or CpG ODNimmunomodulator. Immunization with vaccinia viral constructs results inthe uptake and presentation of viral proteins by DC (Norbury 2002). Indraining lymph nodes, the DC present antigen to naïve CD8⁺ T cells,resulting in T cell activation and the subsequent propagation of animmune response (Norbury 2002). Immunomodulator experiments weredesigned to determine the feasibility of augmenting the response torMVAp53 by addressing both the initiation of the response and itspropagation.

One potent strategy for optimizing tumor vaccines involves manipulatingnegative regulation of T cell responsiveness by using a molecule thatblocks CTLA-4 engagement with ligand, a phenomenon referred to as“CTLA-4 blockade.” CTLA-4 is a cell surface receptor found on T cells.Activation of CTLA-4 leads to inhibition of T cell responses. CTLA-4plays a significant role in regulating peripheral T-cell tolerance byinterfering with T-cell activation through both passive and activemechanisms (Egen 2002). Application of a CTLA-4 blocker in combinationwith cancer vaccines expressing tumor associated autoantigens can, insome cases, result in tumor rejection along with breaking of tolerance,albeit with the concomitant induction of autoimmunity (Espenschied 2003;Hurwitz 2000; van Elsas 1999). In vitro, CTLA-4 blockade lowers theT-cell activation threshold and removes the attenuating effects ofCTLA-4. CTLA-4 blockade also inhibits Treg cell activity in vivo (Read2000). When combined with GM-CSF producing tumor cell vaccines, CTLA-4blockade results in rejection of established poorly immunogenicmelanoma, mammary carcinoma, and prostate carcinoma grafts (Hurwitz1998; Hurwitz 2000; van Elsas 1999). This occurs through a process,which involves breaking tolerance to tumor associated antigens. CTLA-4blocking agents are molecules that specifically bind to the CTLA-4receptor and interfere with the binding of CTLA-4 to itscounter-receptors. A CTLA-4 blocking agent can be a monoclonal orpolyclonal antibody, a fragment of an antibody, a peptide, a smallorganic molecule, a peptidomimetic, a nucleic acid such as interferingRNA (iRNA) or antisense molecule, an aptamer, or any domains from CTLA-4ligands, including members of the B7 family of CTLA-4 ligands, whereinsaid ligands can be preferably synthesized as recombinant solubleproteins capable of binding CTLA-4 present on immune cells and blockingCTLA-4 function. Anti-CTLA-4 antibodies may be generated by immunizing ahost animal with CTLA-4 protein or with cells expressing CTLA-4.Monoclonal antibodies to CTLA-4 (anti-CTLA-4 mAb) can be produced byconventional techniques, namely fusing a hybridoma cell with a mammalianimmune cell that produces anti-CTLA-4 antibody. Mammalian cells used togenerated anti-CTLA-4 mAb may include rat, mouse, hamster, sheep, orhuman cells. Anti-CTLA-4 mAbs may be purified from hybridoma cellsupernatants or from ascites fluid. Anti-CTLA-4 antibodies may be humanantibodies generated using transgenic animals (Bruggemann 1991; Mendez1997) or human immunoglobulin phage display libraries (Winter 1994).Anti-CTLA-4 antibodies also encompasses chimeric and humanized (or“reshaped”) antibodies. Chimeric antibodies to CTLA-4 may be generatedthrough recombinant methods to contain the CTLA-4 binding domain of anon-human antibody and the constant domain of a human antibody.Humanized antibodies to CTLA-4 may be generated by recombinant methodsto contain only the CDR regions of non-human anti-CTLA-4 antibodiesplaced on a human antibody structural framework (Jones 1986; Low 1986).Individual residues within the non-human region may be substituted withresidues from the human antibody framework. Conversely, individualresidues within the human antibody framework may be substituted withresidues from the non-human antibody (Foote 1992). Such substitutionsmay be used to increase the binding capabilities of the humanizedantibody or to decrease the immune response against the antibody.Humanized antibodies to CTLA-4 can be the product of an animal havingtransgenic human immunoglobulin constant region genes. They can also beengineered by recombinant DNA techniques to substitute the C_(H)1,C_(H)2, C_(H)3, hinge domains, or other domains with the correspondinghuman sequence, by methods known in the art.

Oligodeoxynucleotides containing unmethylated CpG(cytosine-phosphate-guanine) motifs are potent immunostimulatory agentsthat can enhance vaccine potency (Krieg 2002). Immune activation by CpGODN initiates with specific binding to the Toll-like Receptor-9 (TLR9)in B cells and plasmacytoid dendritic cells (Krieg 2002). TLR9 ligationin DC results in secondary activation of lymphocyte, macrophage,monocyte, natural killer (NK), and T-cell populations through theelaboration of cytokines generating a T_(H)1 cytokine milieu (Krieg2003). This results in increased NK activity, improved antigenpresentation, and T cell help that can augment both humoral andcell-mediated immune responses. In addition, TLR9 ligation results inthe production of IL-6 by DCs, which helps overcome the suppressiveeffect of CD4⁺ CD25⁺ Treg cells (Pasare 2003). Administration of CpG ODNalone has been shown to exert modest anti-tumor effects in a number ofmurine tumor models (Carpentier 1999; Kawarada 2001; Ballas 2001; Baines2003; Sharma 2003). CpG ODN has been shown to be an effective adjuvantfor a variety of experimental tumor vaccines in mice. It is at least aseffective as Freund's adjuvant, but with higher T_(H)1 activity and lesstoxicity (Chu 1997; Weiner 1997). CpG ODN can enhance the effect ofpeptide (Davila 2000; Stern 2002), protein (Kim 2002), DC (Heckelsmiller2002), idiotype (Baral 2003), and GM-CSF secreting tumor cell vaccines(Sandler 2003). The ability of CpG ODN to prime for TH1 responses andstimulation of NK cells probably accounts for the immunomodulatoractivity in these vaccine approaches and in those described below.

To determine whether administration of a CTLA-4 blocking agent inconjunction with rMVAmup53 vaccination would be beneficial or wouldinduce autoimmune disease, a monoclonal antibody specific to CTLA-4(anti-CTLA-4 mAb) was used. Vaccination with rMVAmup53 and anti-CTLA-4mAb was shown to effect the rejection of established, palpable Meth Atumors. Mice injected with a high dose of Meth A and vaccinated withrMVAmup53 and anti-CTLA-4 mAb (9H10) only after formation of a palpabletumor nodule exhibited complete tumor regression and lasting tumorimmunity. In vivo antibody depletion studies confirmed that thisantitumor effect was primarily CD8⁺, and to a lesser extent CD4⁺,dependent.

To establish that the above results were not tumor specific, vaccinationwith rMVAmup53 and a CTLA-4 blocker immunomodulator was performed onmice injected with 11A-1 or MC-38 tumor cells. 11A-1 is a rapidlygrowing malignant cell line that is poorly immunogenic. MC-38 is a coloncarcinoma cell line. Mice injected with 11A-1 or MC-38 tumor cells andvaccinated 4 days later with rMVAmup53 and anti-CTLA-4 mAb rejectedtheir tumors. Similar results were seen when the anti-CTLA-4 mAb wasreplaced with CpG ODN. The majority of mice treated with rMVAmup53 andCpG ODN did not develop palpable tumors and developed lasting tumorimmunity, rejecting a rechallenge at 60 days.

The potential additive effect of the anti-CTLA-4 mAb and CpG ODNimmunomodulators was examined by administering both immunomodulators inconjunction with rMVAmup53 to 11A-1 injected mice with palpable tumors.Tumor rejection and prolonged survival were observed in the majority ofmice receiving both immunomodulators in conjunction with rMVAmup53. Micethat received only one immunomodulator in conjunction with rMVA, on theother hand, all eventually succumbed to tumor growth. Not only did thecombination of both immunomodulators provide a greater benefit thaneither immunomodulator acting alone, but their combined benefit wasgreater than the simple addition of the effects of the immunomodulators.Similar results were seen in mice bearing MC 38 tumors.

To determine the efficacy of a recombinant MVA containing a human p53sequence, rMVAhup53 was administered to hupki mice injected with4T1(H-2^(d)) cells that had been transfected with human p53. 4T1(H-2^(d)) is a murine breast carcinoma cell line. Mice were vaccinatedwith rMVAhup53 6 days after injection with 4T1 cells, and vaccinatedagain ten days later. During the second vaccination, CpG ODN andanti-CTLA-4 mAb were administered as well. Mice treated with vaccine andboth immunomodulators exhibited a statistically significant improvementin survival.

The above results demonstrate the efficacy of a novel rMVAmup53 orrMVAhup53 cell-free vaccine at eliciting an immune response targetingp53 in a variety of malignant tumor types, as well as the efficacy ofanti-CTLA-4 mAb and CpG ODN as immunomodulators to this vaccine.Accordingly, the present invention provides a composition comprising arecombinant MVA virus engineered to express p53 (rMVAp53). The presentinvention further provides an immunotherapeutic method for eliciting animmune response against a wide range of p53-expressing malignancies byadministering rMVAp53

Introduction of rMVAp53 into a subject can be performed by any procedureknown to those skilled in the art, and is not dependent on the locationof tumor nodules for efficacy or safety. Thus, rMVAp53 can beadministered by intravascular, subcutaneous, peritoneal, intramuscular,intradermal or transdermal injection, to name a few possible modes ofdelivery. rMVAp53 can be prepared as a formulation at an effective dosein pharmaceutically acceptable media, such-as normal saline, vegetableoil, mineral oil, PBS, etc. Therapeutic preparations may includephysiologically tolerable liquids, gel or solid carriers, diluents,adjuvants and excipients. Additives may include bactericidal agents,additives that maintain isotonicity (e.g., NaCl, mannitol), additivesthat maintain chemical stability (e.g., buffers, preservatives) andother ingredients. For parenteral administration, the rMVAp53 may beformulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle.Liposomes or non-aqueous vehicles, such as fixed oils, may also be used.The formulation may be sterilized by techniques known in the art.

The rMVAp53 formulation can be further enhanced with a costimulator,such as a cytokine, tumor antigen, an antigen derived from a pathogen,or any immunomodulator. The costimulator can be any agent that directlyor indirectly stimulates an immune response in combination with therMVAp53, and maybe selected for its ability to modulate APC or T-cellfunction. For example, MVA can be engineered to express GM-CSF, IL-12,or other stimulatory cytokines to produce a costimulator, and thecombination of rMVAp53 and costimulator (here: MVA expressing thestimulatory cytokine) can be introduced into the subject. The treatmentmay be performed in combination with administration of cytokines thatstimulate antigen presenting cells, such as granulocyte-macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3(IL-3), interleukin 12 (IL-12), and others well known in the art. Othercostimulators include cytokine-transduced tumor cells, such as tumorcells transduced with GM-CSF, as well as tumor cells that have beenirradiated and/or treated with a chemotherapeutic agent ex vivo or invivo. Chemotherapeutic or radiotherapeutic agents are further examplesof costimulators. Thus, rMVAp53 can be administered in conjunction witha variety of costimulators known to those of skill in the art.

The formulation is administered at a dose effective to increase theresponse of T cells to antigenic stimulation. The determination of the Tcell response will vary with the condition that is being treated. Usefulmeasures of T cell activity are proliferation, the release of cytokines,including, IL-2, IFNγ, TNFα, etc; T cell expression of markers such asCD25 and CD69; and other measures of T cell activity as known in theart. The dosage of the therapeutic formulation will vary widely,depending upon the stage of the cancer, the frequency of administration,the manner or purpose of the administration, the clearance of rMVAp53from the subject, and other considerations. The dosage administered willvary depending on known factors, such as the pharmacodynamiccharacteristics of the particular agent, mode and route ofadministration, age, health and weight of the recipient, nature andextent of symptoms, concurrent treatments, frequency of treatment, andeffect desired. The dose may be administered as infrequently as weeklyor biweekly, or fractionated into smaller doses and administered daily,semi-weekly, etc., to maintain an effective dosage level.

Generally, a daily dosage of active ingredient can be about 10⁶-10¹¹ IU(infectious units)/kg of body weight. Dosage forms suitable for internaladministration generally contain from about 10⁶ to 10¹² IU of activeingredient per unit. The active ingredient may vary from 0.5 to 95% byweight based on the total-weight of the composition. In some cases itmay be desirable to limit the period of treatment due to excessive Tcell proliferation. The limitations will be empirically determined,depending on the response of the patient to therapy, the number of Tcells in the patient, etc. The number of T cells may be monitored in apatient by methods known in the art, including staining with T cellspecific antibodies and flow cytometry.

In a preferred embodiment of the present invention, rMVAp53 isadministered in conjunction with an immunomodulator, specifically aCTLA-4 blocking agent or a CpG ODN. The combined administration ofrMVAp53 and the CTLA-4 blocking agent anti-CTLA-4 mAb is unexpectedlypotent in producing regression of advanced tumors that are rapidlylethal when left untreated. The same is true of the combinedadministration of rMVAp53 and CpG ODN. Potency is even greater when bothimmunomodulators are administered in conjunction with rMVAp53. Inaddition, the anti-CTLA-4 mAb CpG ODN immunomodulators are nontoxic tothe subject, and capable of generating long lasting immunity to lethalchallenges with tumor cells when administered in conjunction withrMVAp53. As is the case with rMVAp53 alone, introduction of rMVAp53 plusanti-CTLA-4 mAb and/or CpG ODN into a subject can be performed by anyprocedure known to those skilled in the art, and is not dependent on thelocation of tumor nodules for efficacy or safety. Thus, rMVAp53,anti-CTLA-4 mAb, and CpG ODN can be administered by intravascular,subcutaneous, peritoneal, intramuscular, intradermal or transdermalinjection, to name a few possible modes of delivery. rMVAp53,anti-CTLA-4 mAb, and CpG ODN can be administered together, separately,or sequentially, in any order, by the same route of administration or bydifferent routes. rMVAp53 plus anti-CTLA-4 mAb and/or CpG ODN can beprepared as formulations at an effective dose in pharmaceuticallyacceptable media, for example normal saline, vegetable oil, mineral oil,PBS, etc. Therapeutic preparations may include physiologically tolerableliquids, gel or solid carriers, diluents, adjuvants and excipients.Additives may include bactericidal agents, additives that maintainisotonicity, e.g. NaCl, mannitol; and chemical stability, e.g. buffersand preservatives and other ingredients. rMVAmup53 plus anti-CTLA-4 mAband/or CpG ODN may be administered as a cocktail or as single agents.For parenteral administration, anti-CTLA-4 mAb and CpG ODN may beformulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle.Liposomes or non-aqueous vehicles, such as fixed oils, may also be used.The formulation may be sterilized by techniques as known in the art.

The rMVAp53 plus anti-CTLA-4 mAb and/or CpG ODN combination can befurther enhanced with a costimulator such as a cytokine, tumor antigen,or antigen derived from a pathogen. A costimulator can be any agent thatdirectly or indirectly stimulates an immune response in combination withrMVAp53 or in combination with rMVAp53 plus anti-CTLA-4 mAb and/or CpGODN. For example, MVA can be engineered to express GM-CSF, IL-12, orother stimulatory cytokine to produce a costimulator, and thecombination of rMVAp53 and costimulator (here: MVA expressing thestimulatory cytokine), or rMVAp53 plus anti-CTLA-4 mAb and/or CpG ODNand costimulator can be introduced into the subject. The treatment maybe performed in combination with administration of cytokines thatstimulate antigen presenting cells, such as granulocyte-macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3(IL-3), interleukin 12 (IL-12) and others well known in the art. Othercostimulators include cytokine-transduced tumor cells such as tumorcells transduced with GM-CSF, or tumor cells that have been irradiatedand/or treated with a chemotherapeutic agent ex vivo or in vivo.Chemotherapeutic or radiotherapeutic agents are further examples ofcostimulators. Thus, rMVAp53 either alone or in combination withanti-CTLA-4 mAb and/or CpG ODN can be administered in conjunction with avariety of costimulators known to those of skill in the art.

The dosage of the therapeutic formulation will vary widely, dependingupon the stage of the cancer, the frequency of administration, themanner or purpose of the administration, and the clearance of rMVAp53,anti-CTLA-4 mAb, and CpG ODN from the subject, among otherconsiderations. The dosage administered will vary depending on knownfactors, such as the pharmacodynamic characteristics of the particularagent, mode and route of administration, age, health and weight of therecipient, nature and extent of symptoms, concurrent treatments,frequency of treatment and effect desired. The dose may be administeredas infrequently as weekly or biweekly, or fractionated into smallerdoses and administered daily, semi-weekly, etc. to maintain an effectivedosage level.

Generally, a daily dosage of active ingredient (antibody) can be about0.1 to 100 mg/kg of body weight. Dosage forms suitable for internaladministration generally contain from about 0.1 mg to 500 mgs of activeingredient per unit. The active ingredient may vary from 0.5 to 95% byweight based on the total weight of the composition. In some cases itmay be desirable to limit the period of treatment due to excessive Tcell proliferation. The limitations will be empirically determined,depending on the response of the patient to therapy, the number of Tcells in the patient, etc. The number of T cells may be monitored in apatient by methods known in the art, including staining with T cellspecific antibodies and flow cytometry. The formulation is administeredat a dose effective to increase the response of T cells to antigenicstimulation. The determination of the T cell response will vary with thecondition that is being treated. Useful measures of T cell activity areproliferation, the release of cytokines, including. IL-2, IFNγ, TNFα,etc; T cell expression of markers such as CD25 and CD69; and othermeasures of T cell activity as known in the art.

The present invention further provides a kit that will allow the artisanto prepare an immunotherapeutic regimen for eliciting an immune responseagainst a p53-expressing malignancy. An example of a kit comprisesrMVAp53, a CTLA-4 blocking agent and/or a CpG ODN, and instructions forusing these compounds to elicit an immune response against ap53-expressing malignancy in a subject. The kit may further comprise oneor more pharmaceutically acceptable carriers. When administered, thecompositions of the kit are administered in pharmaceutically acceptablepreparations. The terms administration, administering, and introducingrefer to providing the compositions of the invention as a medicament toan individual in need of treatment or prevention of a p53-expressingmalignancy. This medicament, which contains compositions of the presentinvention as the principal or active ingredients, can be administered ina wide variety of therapeutic dosage forms in the conventional vehiclesfor topical, oral, systemic, local, and parenteral administration. Thus,the kits of the invention provide compositions for parenteraladministration that comprise a solution of the compositions dissolvedor-suspended in an acceptable carrier, preferably an aqueous carrier.The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, including sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, and many others. Actual methods for preparingcompounds for parenteral administration will be known or apparent tothose skilled in the art and are described in more detail in, forexample, Remington: The Science and Practice of Pharmacy (“Remington'sPharmaceutical Sciences”) Gennaro AR ed. 20^(th) edition, 2000: Williams& Wilkins PA, USA, which is incorporated herein by reference.

Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents. All the preparationsof the invention are administered in effective amounts. An effectiveamount is that amount of a pharmaceutical preparation that alone, ortogether with further doses, stimulates the desired response. In thecase of treating cancer, the desired response is inhibiting theinitiation or progression of the cancer, or producing regression of thecancer. This may involve only slowing the progression of the diseasetemporarily, although more preferably, it involves halting theprogression of the disease permanently. These desired responses can bemonitored by routine methods or can be monitored according to diagnosticmethods of the invention discussed herein. It is believed that doses ofimmunogens ranging from 10⁴ IU/kilogram to 10¹¹ IU/kilogram, dependingupon the mode of administration, would be effective. The preferred rangeis believed to be between 10⁶ IU and 10⁹ IU per kilogram. The absoluteamount will depend upon a variety of factors, including the combinationselected for administration, whether the administration is in single ormultiple doses, and individual patient parameters including age,physical condition, size, weight, and the stage of the disease. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. Unless otherwise specified, general cloning procedures, suchas those set forth in Sambrook et al., Molecular Cloning, Cold SpringHarbor Laboratory (2001), Ausubel et al. (Eds.) Current Protocols inMolecular Biology, John Wiley & Sons (1986, 2000) are used. One skilledin the art may develop equivalent means or reactants without theexercise of inventive capacity and without departing from the scope ofthe invention.

It will be understood that many variations can be made in the proceduresherein described while still remaining within the bounds of the presentinvention. Likewise, it is understood that, due to the degeneracy of thegenetic code, nucleic acid sequences with codons equivalent to thosedisclosed will encode functionally equivalent or identical proteins asdisclosed herein. It is the intention of the inventors that suchvariations are included within the scope of the invention.

EXAMPLES

Materials and Methods

Animals

Female 6-8 week old Balb/c, C57BL/6, B6.129S2-IL6^(tm1Kopf) (IL-6^(−/−),and IFN-γ knock out (IFNγ^(KO)) mice on the Balb/c background wereobtained from The Jackson Laboratory (Bar Harbor, Me.). TLR9^(−/−) micewere a kind gift from Dr. Shizuo Akira (Osaka University, Osaka, Japan).Mice were maintained in a specific pathogen-free environment. Allstudies were approved by the Research Animal Care Committee of the Cityof Hope National Medical Center, and performed under the AAALACguidelines.

Cell Lines

CV-1 (Kit 1965), TK⁻ (Berson 1996), and Baby Hamster Kidney cells(BHK-21) (Macpherson 1962) were purchased from American Type CultureCollection (ATCC) (Manassas, Va.), and grown in MEM supplemented withnon-essential amino acids, L-glutamine, and 10% FCS. 11A-1 (Selvanayagam1995) was a kind gift from Dr. R. L. Ullrich (University of TexasMedical Branch, Galveston, Tex.). Hek 293 cells and p53null 10.1 cellswere kind gifts from Dr. K. K. Wong and Dr. Susan Kane (City of HopeNational Medical Center, Duarte, Calif.). MC-38 (Tan 1976) was a kindgift from Dr. S. A. Rosenberg (National Cancer Institute, Bethesda, Md.Meth A sarcoma cells (Meth A) (DeLeo 1977) were a kind gift from Dr. L.J. Old (Memorial Sloan-Kettering Cancer Center, New York, N.Y.). Meth Awas passaged as an ascitic tumor. Cells were harvested, counted andwashed with PBS prior to use. The characteristics of the Meth A, 11A-1,and MC-38 tumor cell lines are summarized in the following table:

P53 mutation Cell line Tumor MHC Background position(s) Meth AFibrosarcoma H-2^(d) 132, 168, 234 11A-1 Mammary cell H-2^(d) 173carcinoma MC-38 Colon carcinoma H-2^(b) 242

Antibodies

Anti-CD4 (GK1.5) (Dialynas 1983) and anti-NK1.1 (PK136) (Koo 1984) werepurchased from ATCC. Anti-CD8 (H35) (Miconnet 2001) and anti-CTLA-4 mAb(9H10) (Krummel 1995) were kind gifts from James P. Allison (Universityof California, Berkeley, Calif.). Antibodies were produced using aCELLine Device (BD Biosciences, Bedford, Mass.). IgG antibodies werepurified by absorbance over protein G-Sepharose (Amersham, Uppsala,Sweden) followed by elution with 0.1 M Glycine-HCl, pH 2.7. The productwas then dialyzed against phosphate-buffered normal saline (PBS) andconcentrated using a Centriplus centrifugal filter device (Millipore,Bedford, Mass.). Control Syrian Hamster IgG was obtained from JacksonImmuno Research (West Grove, Pa.).

Viral Constructs

rMVA Expressing Murine p53 (rMVAmup53):

Wild type MVA (wtMVA) was obtained from Dr. Bernard Moss and Dr. LindaWyatt (National Institutes of Health Bethesda, Md.). wtMVA stocks forthe generation of recombinant MVA (rMVA) containing mup53 are propagatedon specific pathogen free chicken embryo fibroblasts (SPF/CEF). ThewtMVA stock is titrated by immunostaining, aliquoted, and stored at −80°C.

Murine p53 (mup53) is analogous to human p53, with 80% sequence homology(Halevy 1991; Sukumar 1995). The mRNA coding sequence for full-lengthwild type mup53 is shown in SEQ ID NO: 1. The level of homology betweenmurine and human p53 makes the murine system an excellent preclinicalmodel for evaluating immunologic approaches for overcoming tolerance top53. rMVA expressing murine p53 was generated by homologousrecombination of wtMVA and a pMCO3 insertion vector containing a murinep53 insert, as described in Espenschied 2003. The entire cDNA of murinewild type p53 was amplified by PCR of mRNA obtained from murinesplenocytes. The murine p53 PCR product was ligated into the cloningsite of the MVA expression vector pMCO3 (also obtained from Dr. Moss andDr. Wyatt). This vector contains sequences that insert into deletion IIIof the MVA genome, and also contains the gus (E. coli B-glucuronidase)operon for screening purposes (Ourmanov 2000). Generation of recombinantMVA was achieved on monolayers of BHK-21 cells (Espenschied 2003).Briefly, BHK-21 cells were transfected with 20 μg of plasmid DNA usingLipofectin (Invitrogen, Carlsbad, Calif.) and infected with wtMVA at anmoi of 0.01. The infected cells were incubated for 48 hours, thenharvested, pelleted, and subjected to 3 cycles of freeze/thaw andsonication to lyse the cells. rMVA virus expressing murine p53(rMVAmup53) was screened for gus expression by adding X-GlcA(5-Bromo-4-Chloro-3-Indolyl B-D-Glucuronide, Sigma-Aldrich, St Louis,Mo.). After 10 rounds of purification, the rMVAmup53 was expanded onBHK-21 monolayers. The rMVAmup53 titer was determined by immunostaininginfected cultures using the Vectastain Elite ABC Kit (VectorLaboratories, Burlingame, Calif.).

rMVA Expressing Human p53 (rMVAhup53):

Two different constructs of rMVA expressing human p53 (rMVAhup53) weremade. The mRNA sequence encoding full-length wild type hup53 is shown inSEQ ID NO: 2. The first was made using the pLW51 insertion plasmid,while the second was made using the pLW22 insertion plasmid. wtMVA usedto make the first construct was propagated on SPF/CEF. wtMVA used tomake the second construct was propagated on BHK-21 (BHK) cells. wtMVAstock was titrated by immunostaining, aliquoted, and stored at −80° C.

pLW51 was used as the insertion plasmid for generating the firstrMVAhup53 construct. pLW51 has four important features. First, itcontains MVA flanking regions of deletion III that allow it to insertinto the deletion III region of MVA via homologous recombination.Second, it contains a color screening marker gene, β-glucoronidase(gus), under control of a vaccinia promoter called P₁₁. Third, itcontains two direct repeats composed of MVA sequence (designated as DR1and DR2) flanking the gus screening marker gene to allow the gus gene tobe removed from recombinant MVA. Finally, it contains two vacciniapromoters (P_(SYN) and P_(7.5)) and two multiple cloning sites (MCS),permitting the insertion of two separate foreign genes under the controlof the P_(SYN) and P_(7.5) promoters. The first MCS is behind anearly/late P_(SYN) promoter, while the second MCS uses an early/lateP_(mH5) promoter. This enables the elimination of the gus marker genethrough recombination via a set of direct repeats, which flank it. Thegeneration of the initial rMVA stock is done on CEF utilizing methodsthat were previously described for BHK cells, with modifications toaccount for good laboratory practice (GLP) conditions. About 40-50 fociare pulled from the first rounds of screening to ensure that a correctrecombinant will be found, after which 5-10 are pulled in eachsubsequent round. After each round of selection, either immunostainingor immunofluorescence is performed on each plug to make sure that theplug is expressing the hup53 gene. To achieve a virus that will bedeleted of the bacterial gene marker, purified rMVA expressing hup53 isplated at low dilution in 24 well plates. Wells that do not have a colorreaction demonstrating the gus gene are further analyzed for thepresence of the hup53 gene product. This is accomplished by antibodystaining using conditions that allow recovery of the virus from thecells. Those wells that exhibit hup53 immunostaining in the absence of acolor reaction are further propagated and confirmed to be the correctphenotype. A portion of the viral plug pulled from the final round ofscreening absent the gus marker is expanded in a 100 mm tissue culturedish of CEF. This is followed by DNA extraction and PCR analysis(discussed below).

pLW22 was used as the insertion plasmid for generating the secondrMVAhup53 construct. pLW22 has MVA flanking regions that allow it toinsert into MVA via homologous recombination. It also has a colorscreening marker gene, β-galactosidase. To obtain DNA encoding wt hup53,pHp53B plasmid in E. coli was obtained from the ATCC (#57254). Hup53 wasamplified from the pHp53B plasmid using the forward primer of SEQ ID NO:3 and the reverse primer of SEQ ID NO: 4., Amplified wt hup53 DNA wasinserted into the pLW22 vector between restriction sites Pme-1 andAsc-1, generating pLW22-hup53. The plasmid sequence of pLW22-hup53 isshown in SEQ ID NO: 5.

Generation of rMVA was achieved on monolayers of BHK cells. BHK cellswere transfected with 20 μg of plasmid DNA using Lipofectin (Invitrogen,Carlsbad, Calif.), and infected with wtMVA at an moi of 0.01. Theinfected cells were incubated for 48 hours, then harvested, pelleted,and subjected to three cycles of freeze/thaw and sonication to lyse thecells. rMVA expressing hup53 was screened for β-gal expression by addingpresence of Bluo-gal™ substrate (Sigma-Aldrich, St Louis, Mo.)(Chakrabarti 1985). After 10 rounds of purification, the rMVAhup53 wasexpanded on BHK monolayers. The rMVA titer was determined byimmunostaining infected cultures using the Vectastain Elite ABC kit(Vector Laboratories, Burlingame, Calif.).

For both constructs, a standard DNA extraction is performed. Ethanolprecipitation of 50 μL of the cell lysate resulted in enough DNA to runa PCR reaction to assure the absence of contaminating wtMVA. One set ofPCR primers are designed outside the flanking regions of therecombination site for which the gene has been inserted. The presence ofunmodified wtMVA sequence will generate a 500 bp PCR product, whereasthe insertion of the sequence containing hup53 has a much largerfragment (>6 kb), which is usually difficult to amplify under standardPCR conditions. A second set of PCR primers are designed to amplify asequence within the hup53 insert. The presence of the hup53 insert willgenerate a 300 bp PCR product. The PCR samples are run on a 1% agarosegel and analyzed to determine if additional screenings are necessary toremove any remaining wtMVA. Examples of purified MVA containing humanp53 have been shown to be absolutely homogenous (FIG. 1).

rMVA Expressing pD65 (rMVApp65):

rMVA expressing pp65 (rMVApp65), a CMV tegument protein, was constructedusing techniques similar to those used to construct rMVAmup53 (Gibson).

rVV Expressing Murine P53 or DD65:

Recombinant Western Reserve strain Vaccinia Virus expressing murine wildtype p53 or pp65 (rVVp53, rVVpp65) was constructed using publishedtechniques (Diamond 1997).

rAd Expressing Murine p53:

Recombinant adenovirus expressing wild type murine p53 (rAd-mup53) wasconstructed using the pAd Easy system (He 1998). Both pAd Track-CMV andpAd Easy-1 plasmids were kindly provided by Dr. Bert Vogelstein (JohnsHopkins Oncology Center, Baltimore, Md.). Wild type murine p53 cDNA wascloned into the Bgl II and Xba I site of a pAd Track-CMV shuttle vectorcontaining green fluorescent protein (GFP) with a CMV promoter (p53-pAdTrack-CMV). The p53-pAd Track-CMV was cotransformed into BJ5183 cellswith the pAd Easy-1 plasmid to generate the p53 recombinant adenoviralconstruct by homologous recombination. The presence of the p53 gene inthe recombinants was confirmed by DNA sequencing. The p53 recombinantadenoviral construct was cleaved with Pac I and transfected into HEK-293cells. rAd-mup53 was harvested 5 days after transfection and p53 proteinexpression was confirmed by western blot. The adenovirus was expanded onHEK-293 cells and purified by cesium chloride gradient. The purifiedvirus was dialyzed in PBS, titered on HEK-293 cells, and stored at −80°C. in 20% glycerol.

Oligodeoxynucleotides (ODN)

Synthetic ODN 1826 with CpG motifs (SEQ ID NO: 6) and non-CpG 6DN 1982(SEQ ID NO: 7) (Moldoveanu 1998), were synthesized withnuclease-resistant phosphorothioate backbones by Trilink (San Diego,Calif.). The Na⁺ salts of the ODNs were resuspended at 5 mg ml⁻¹ in 10mM Tris (pH 7.0) 1 mM EDTA and stored as 50 μl aliquots at −20° C.before dilution in aqueous 0.9% sodium chloride solution prior toinjection.

Example 1 Expression of Murine p53 Protein By rMVAmup53

Expression of murine p53 protein following infection with rMVAmup53 wasanalyzed to determine the fidelity and extent of its expression fromrecombinant virus. Lysates were prepared from BHK or HEK 293 cellsinfected with rMVAmup53 and subjected to SDS-PAGE and Western blotting.Standard Western Blotting techniques were performed using an ECL WesternBlot Kit (Amersham Pharmacia Biotech, England). The samples wereincubated with a purified mouse anti-p53 monoclonal antibody, PAb 122(Gurney 1980), followed by incubation with a peroxidase labeled goatanti-mouse secondary antibody provided in the ECL Western Blot kit.Western blot analysis of BHK cells infected with rMVAmup53 demonstratesabundant p53 expression (FIG. 2). The remarkable level of expressionexhibited by rMVAmup53 compared to other viral and cellular formsdemonstrates its usefulness in vaccination-protocols. As shown in FIG.1, the volume on the rMVAmup53 lane is between 80-160 fold less thanwhat was applied to the gel in the other lanes, yet the intensity of theband is several fold higher. This demonstrates a very high level of p53expression by rMVAmup53. Meth A cells were used as a positive controland BHK cells infected with HCMV IE1 exon 4 rMVA were used as negativecontrols. Meth A is a Balb/c derived, tumorigenic3-methylcholanthrene-induced sarcoma that over-expresses mutated p53. A53 kilodalton band was observed in both the p53 overexpressing Meth Asarcoma and the rMVAmup53 infected BHK cells (FIG. 1). This contrastswith the absence of 1-5 detectable p53 expression in the HCMV IE1 exon4-rMVA infected BHK cells. Strong p53 expression was also observed byfluorescence microscopy in BHK cells infected with rMVAmup53 (data notshown).

Example 2 In vitro Generation of a p53-Specific CTL Response ByrMVAmup53

Vaccination of mice with rMVA expressing viral and tumor associatedantigens results in enhanced antigen specific CTL responses. One goal ofthis example was to determine if vaccination with rMVAmup53 could breakp53 tolerance, resulting in the generation of p53-specific CTL. Micewere vaccinated i.p. with 5×10⁷ pfu of either rMVAmup53 or rMVApp65.After two weeks, spleens were harvested and disassociated, andsplenocytes were washed and counted.

Splenocytes were restimulated in vitro for 6 days with syngeneic LPSblasts infected with rAd-mup53 or rMVAmup53. Na-⁵¹CrO₄-labeled targetcells that overexpress wt p53 were added to 96 well plates with theeffectors, in triplicate, at various effector to target ratios, in 200μl of complete medium. The plates were incubated for 4 hours at 37° C.,and the supernatant was harvested and analyzed. Percent specific lysiswas calculated using the formula: percent specific release=(experimentalrelease—spontaneous release)/(total release−spontaneous release)×100.Splenocytes vaccinated with rMVAmup53 recognized and lysed target cellsthat overexpressed wt p53 (FIG. 3). In contrast, splenocytes from micevaccinated with rMVApp65, which stimulates a vigorous pp65 specific CTLresponse, did not recognize the p53 over-expressing targets (FIG. 3B),demonstrating the specificity of the lymphocyte response. rMVAmup53vaccination can also stimulate CTL recognition of a cell line bearingmutated p53, Meth A. Restimulated splenocytes vaccinated with rMVAmup53recognized mutant p53 over-expressing Meth A cells, but splenocytesvaccinated with rMVAmup53 did not (FIG. 3 c).

Example 3 In Vivo rMVAmup53 Tumor Challenge Experiments

Since a single vaccination with rMVAmup53 resulted in enhanced CTLresponses, there was sufficient justification to examine the effect ofrMVAmup53 vaccination on the growth of tumor cells in vivo.

Statistical Methods

For experiments where the growth rate of some tumors necessitated earlysacrifice, growth curves were compared by the time to a fixed size usinga logrank test. Contrasts of single groups to all others were conductedafter a single omnibus test. For cell depletion experiments, all micewere followed for a fixed amount of time, and final tumor size wascompared by the Wilcoxon rank-sum test, after a significantKruskal-Wallis test if there were more than two groups. For survivalexperiments, a logrank test was used.

rMVAmuD53 vs. Meth A Cells

Six-week-old female Balb/c mice were injected by subcutaneous (s.c.)route in the left flank with 5×10⁵ Meth A cells. Mice injected s.c. withMeth A cells develop a rapidly growing fibrosarcoma that kills themajority of mice within 21 days (FIG. 3). On day 3, the mice werevaccinated with 5×10⁷ pfu of rMVAmup53 by intraperitoneal (i.p.)injection. Negative control mice were injected with 5×10⁷ rMVApp65 orPBS. The s.c. tumors were measured twice weekly in three dimensions withcalipers. Tumors in rMVAmup53 treated animals grew at a much slower ratethan those in control animals. At 14 days, the mean s.c. tumor volumefor the rMVAmup53 treated group (n=16) was dramatically lower than boththe rMVApp65 (n=16) and PBS (n=112) controls (22 mm³ versus 348 mm³,p<0.001 and 22 mm³ versus 252 mm³, p<0.001 by Student's t-test).Survival of rMVAmup53 treated animals was also significantly prolongedcompared to either control group (FIG. 4). 12 of the 16 rMVAmup53immunized mice failed to develop tumors entirely. The 12 tumor freerMVAmup53 treated animals were re-challenged at day 52 with 5×10⁵ Meth Atumor cells. All animals remained tumor free for the duration of a 30day observation period (data not shown).

rMVAmup53 Plus Anti-CTLA-4 mAb vs. Meth A Cells

One potent strategy for optimizing tumor vaccines involves manipulatingnegative regulation of T cell responsiveness using an antibody thatblocks CTLA-4 engagement with ligand. This phenomenon has been referredto as CTLA-4 blockade. Application of anti-CTLA-4 mAb in combinationwith cancer vaccines expressing tumor associated autoantigens, in somecases, results in tumor rejection along with breaking of tolerance andinduction of autoimmunity. Therefore, mAb specific to CTLA-4 was addedto rMVAmup53 vaccination to determine whether it would synergize andaugment the anti-tumor activity against Meth A in vivo. A more rigoroustumor model was designed in order to overcome the potent antitumoreffect of CTLA-4 blockade alone. Six-week-old Balb/c mice were injecteds.c. in the left flank with 10⁶ Meth A cells rather than 5×10⁵ Meth Acells, and treatment was postponed until a palpable tumor nodule wasidentified (Day 6). This more rigorous model overcame the effect of theCTLA-4 blockade, producing a rapidly lethal tumor in the majority ofmice despite anti-CTLA-4 mAb treatment (FIG. 5). On day 7, mice wereinjected i.p. with 5×10⁷ pfu of rMVAmup53. Controls were the same asabove. Anti-CTLA-4 mAb antibody or control hamster Ab were injected i.p.on days 6, 9, and 12 at 100, 50 and 50 μg dose, respectively. 11 of the14 mice immunized with rMVAmup53 plus anti-CTLA-4 mAb rejected tumors,resulting in tumor free survival for the duration of the 60 dayobservation period (FIG. 5). By contrast, mice treated with rMVApp65 andcontrol antibody died rapidly of progressive tumor (FIG. 5) as did PBStreated controls (data not shown). The 11 tumor-free rMVAmup53 plusanti-CTLA-4 mAb treated mice also rejected a re-challenge with 10⁶ MethA tumor cells at 60 days, and remained tumor free for the duration of a30 day observation period (data not shown).

rMVAmuD53 Plus Anti-CTLA-4 mAb vs. 11A-1-Cells

Six-week-old Balb/c mice were injected s.c. in the left flank with 2×10⁶11A-1 cells. 11A-1 is a rapidly growing malignant cell line that ispoorly immunogenic. Mice vaccinated with 10⁶ irradiated 11A-1 tumorcells failed to reject a subsequent challenge with 11A-1 (data notshown). Anti-CTLA-4 mAb or the control hamster antibody was injectedi.p. on days 4, 7, and 10 at 100, 50, and 50 μg/dose, respectively. Onday 5, mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53,5×10⁷ MVApp65, or PBS. s.c. tumors were measured twice weekly in threedimensions with calipers. Mice vaccinated with rMVAmup53 plusanti-CTLA-4 mAb rejected their tumors (FIG. 6). Animals treated withanti-CTLA-4 mAb alone or with a control MVA vaccine developed rapidlyprogressing lethal tumors (p0.00044, comparing rMVAmup53 withanti-CTLA-4 mAb

rMVAmup53 Plus Anti-CTLA-4 mAb vs. MC-38 Cells

Six-week-old C57BL/6 mice, TLR9^(−/−), or IL-6^(−/−) mice were injecteds.c. in the left flank with 1×10⁶ MC-38 cells. Anti-CTLA-4 mAb or thecontrol hamster antibody was injected i.p. on days 4, 7, and 10 at 100,50, and 50 μg/dose, respectively. On day 5, mice were vaccinated i.p.with either 5×10⁷ pfu of rMVAmup53, 5×10⁷ rMVApp65, or PBS. s.c. tumorswere measured twice weekly in three dimensions with calipers. Micevaccinated with rMVAmup53 plus anti-CTLA-4 mAb rejected their tumors,while those treated with anti-CTLA-4 mAb alone or with a control MVAvaccine developed rapidly progressing tumors (p=0.0001, comparingrMVAmup53 with anti-CTLA-4 mAb to control groups) (FIG. 7).

rMVAmup53 Plus CpG ODN vs. 11A-1 Cells

CpG ODN treatment has been shown to be an effective immunomodulator in anumber of experimental tumor vaccine models (Krieg 2002). Mice werechallenged with 11A-1 tumor as above. 15 nmoles of CpG ODN or thenon-CpG ODN control were injected i.p. on days 4, 9, and 14. On day 5,the mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53, 5×10⁷rMVApp65, or PBS. The s.c. tumors were measured twice weekly in threedimensions with calipers. While rMVAmup53 and CpG ODN each separatelyresulted in minimal attenuation of tumor growth, all animals developedprogressively lethal tumors. The combination of CpG ODN and rMVAmup53vaccination resulted in significantly diminished tumor outgrowth(p=0.00002) (FIG. 8). 6 of the 8 animals treated with rMVAmup53 plus CpGODN did not develop palpable tumors and developed lasting tumorimmunity, rejecting a rechallenge with 11A-1 at 60 days (data notshown).

rMVAmup53 Plus CpG ODN vs. Meth A Cells

A pattern of tumor rejection similar to that for 11A-1 was seenfollowing treatment of early established Meth A tumors in Balb/c mice(p=0.0015) (FIG. 9).

rMVAmuD53 Plus CpG ODN vs. MC-38 Cells

To demonstrate that the immunomodulator effect of CpG ODN on rMVAmup53vaccination is not strain specific, the vaccination strategy wasrepeated in C57BL/6 mice bearing early established MC38 colon cancers.Vaccination with rMVAmup53 plus CpG ODN resulted in significantsuppression of tumor growth (p=0.0004) (FIG. 10).

rMVAmup53 Plus Anti-CTLA-4 mAb Plus CpG ODN vs. 11A-1 Cells

A more rigorous tumor model was designed to evaluate the potentialadditive effects of CpG ODN and anti-CTLA-4 mAb on rMVAmup53vaccination. Six-week-old Balb/c mice were injected s.c. in the leftflank with 2×10⁶11A-1 cells and followed for two weeks until palpabletumors were present. Anti-CTLA-4 mAb or the control hamster antibody wasinjected i.p. on days 14, 17, and 20, at 100, 50, and 50 μg/dose,respectively. 15 nmoles of CpG ODN was injected i.p. on days 14, 19, and24. On day 15, the mice were vaccinated i.p. with either 5×10⁷ pfu ofrMVAmup53, 5×10⁷ MVApp65, or PBS.

rMVAmup53 vaccination combined with either anti-CTLA-4 mAb or CpG ODNimmunomodulators resulted in prolonged survival, but all animalseventually succumbed to progressive tumor growth. The combination ofanti-CTLA-4 mAb and CpG ODN administration with rMVAmup53 vaccinationresulted in tumor rejection and prolonged survival in the majority oftreated animals (FIG. 11). The combination of anti-CTLA-4 mAb and CpGODN provides better immunomodulator activity than either CpG ODN alone(p=0.02) or anti-CTLA-4 mAb alone (p=0.01). The effect of combinedanti-CTLA-4 mAb and CpG ODN administration provides a greater benefit interms of survival at 60 days than the simple addition of the effects ofboth immunomodulators separately.

rMVAmup53 Plus Anti-CTLA-4 mAb Plus CpG ODN vs. MC-38 Cells

A similar pattern was seen in C57BL/6 mice bearing MC 38 tumors (FIG.12). C57BL/6 mice bearing MC-38 tumors were treated with rMVAmup53 plusa combination of anti-CTLA-4 mAb and CpG ODN as described above for11A-1. In this tumor model, the combination of anti-CTLA-4 mAb and CpGODN also provided better immunomodulator activity than either CpG ODNalone (p=0.002) or anti-CTLA-4 mAb alone (p=0.001). The combined effectin both tumor models is not simply a dose additive effect, as the CpGODN and anti-CTLA-4 mAb were both already administered at doses ofmaximal efficacy. The striking increases in activity found when bothimmunomodulators are used together in at least two different tumorssuggests that further investigation of the combined modality iswarranted in humans.

Example 4 Cellular Requirements for Anti-CTLA-4 mAb and CpG ODNImmunomodulator Effect

To determine the cellular requirements for the immunomodulator effect ofanti-CTLA-4 mAb and CpG ODN, Balb/c mice were depleted of CD4⁺, CD8⁺, orNK cells prior to vaccination. Depletion was accomplished by i.p.injection of 200 μg of CD4⁺, CD8⁺, or NK1.1 cell specific mAbs, or acontrol mAb. Injections were given on days —1, 1, 3, 4, 6, 8, and 15,with a maintenance dose every 7 days until the termination of theanimals. This regimen was shown to deplete (>95%) Balb/c mice ofCD4^(+, CD)8⁺, or NK 1.1 cells based on flow cytometry of peripheralblood from treated animals (data not shown).

The cellular requirements for the immunomodulator effect of CTLA-4blockade on rMVAmup53 vaccination were evaluated using the Meth A tumormodel in Balb/c mice. Mice depleted of CD8⁺ T cells or CD4^(+ and CD)8⁺T cells simultaneously develop rapidly lethal tumors. These tumors areresistant to vaccination with rMVAmup53 and anti-CTLA-4 mAb. Incontrast, CD4⁺ T cell depletion resulted in only a partial abrogation ofresponse to the vaccine. NK1.1 cell depletion had little effect on theability of vaccinated mice to reject Meth A (FIG. 13 a). Results werethe same when the depleting mAbs were administered after vaccine andanti-CTLA-4 mAb treatment (data now shown). Similar results were alsoobtained when the 11A-1 tumor model was used rather than the Meth Atumor model. The therapeutic effect of rMVAmup53, and anti-CTLA-4 mAbcould be eliminated by administering depleting doses of anti-CD8⁺ mAb(p=0.004) (FIG. 15). The antitumor effect was partially blocked by theadministration of depleting anti-CD4⁺ mAb (p=0.008), and unaffected bythe administration of an NK depleting mAb. These results show that theimmunomodulator effect of anti-CTLA-4 mAb is entirely dependent on CD8⁺cells, partially dependent on CD4⁺ cells, and not dependent at all on NKcells (Espenschied 2003).

The cellular requirements for the immunomodulator effect of CpG ODN onrMVAmup53 vaccination were evaluated using Balb/c mice with four-dayestablished 11A-1 tumors. As with anti-CTLA-4 mAb, the immunomodulatoreffect of CpG ODN on MVAmup53 vaccination could be completely abrogatedby the administration of depleting CD8⁺ mAb (p=0.004) (FIG. 14).However, unlike anti-CTLA-4 mAb, the immunomodulator effect of CpG ODNwas unaffected by CD4⁺ depletion, while depletion of NK cells partiallyabrogated the vaccine effect (p=0.007, comparing NK to CD4⁺ and controlantibody depletions). The difference in cellular requirements for CD4⁺and NK between anti-CTLA-4 mAb and CpG ODN is striking, because bothimmunomodulators cause equivalent levels of rejection. These resultssuggest that the two immunomodulators act through differing immunologicmechanisms. This information, combined with the data regarding theeffects of combined anti-CTLA-4 mAb/CpG ODN administration on rMVAmup53,suggest a synergistic effect by the two immunomodulators on tumorgrowth.

Contribution of IFN-γ

The contribution of IFN-γ secretion to the effect of CTLA-4 blockade andrMVAmup53 vaccination was evaluated in IFNγ^(KO) mice. Both unvaccinatedmice and mice vaccinated with rMVApp65 and anti-CTLA-4 mAb developedlethal tumors at a rate similar to that seen in normal Balb/c mice (FIG.13 b). 3 of the 5 IFNγ^(KO) mice that were vaccinated with rMVAmup53 andanti-CTLA-4 mAb developed lethal tumor growth, confirming a contributionof IFN-γ to the vaccine/CTLA-4 blockade effect.

Contribution of TLR 9

The cell subset depletion studies suggest that the mechanism ofimmunomodulator activity of CTLA-4 blockade and CpG ODN is different.CpG ODN activity results from the stimulation of B-cells andplasmacytoid dendritic cells through an interaction with the TLR9receptor (Chu 1997). CpG treatment causes a bias towards the TH1cytokine milieu and stimulation of NK cell proliferation, which mayaccount for the partial effect on tumor rejection. To further delineatethe divergent pathways involved in the CpG ODN and CTLA-4 blockadeimmunomodulator effects, MC-38 tumor challenge experiments wereconducted in TLR9^(−/−) mice. TLR9^(−/−) mice fail to immunologicallyrespond to CpG ODN administration (Hemmi 2000). As expected, TLR9^(−/−)mice bearing early established MC-38 tumors failed to immunologicallyrespond to CpG ODN and rMVAmup53 vaccination (FIG. 16). In contrast,inclusion of anti-CTLA-4 mAb with rMVAmup53 vaccination resulted intumor rejection in TLR9^(−/−) mice (p=0.0009) that was similar to thatseen in wt C57BL/6 mice (FIG. 16, FIG. 7).

Contribution of IL-6

Both CpG ODN and CTLA-4 blockade inhibit CD25⁺ CD4⁺ suppressor orregulatory T cells (Treg), and this effect may contribute to theirimmunomodulator activity in the described tumor models. Blocking CTLA-4is thought to have a direct inhibitory affect on Tregs, most of whichconstitutively express CTLA-4 (Read 2000). In contrast, CpG ODN inhibitsTreg activity through the secretion of IL-6 by DC (Pasare 2003). Toevaluate the role of IL-6 on the CpG ODN and anti-CTLA-4 mAbimmunomodulator effects, tumor challenge experiments were conducted inIL-6^(−/−) mice. IL-6^(−/−) mice bearing early established MC-38 tumorsfailed to immunologically respond to rMVAmup53 vaccination with CpG ODNby rejecting tumor (FIG. 17). This suggests that CpG ODN could bemediating its immunomodulator effects, at least in part, through theIL-6 dependent pathway of Treg cell inhibition. In contrast, anti-CTLA-4mAB inclusion with rMVAmup53 vaccination resulted in tumor rejection inIL-6^(−/−) mice (p=0.02) to an extent similar to that seen in wt C57BL/6mice (FIG. 17, FIG. 7).

Example 5 Expression of Human p53 By rMVAhup53

BHK cells were infected with purified rMVAhup53. Expression of hup53 wasmeasured at 24 and 48 hours, and analyzed by Western blot andimmunohistochemistry. The infected rMVAhup53 cells demonstrated vigorousexpression of hup53 at both time periods (FIG. 18).

Example 6 In vivo rMVAhu53 Tumor Challenge Experiments

Hupki mice, a novel murine knock-in model expressing human p53, wereobtained from Dr. Monica Hollstein (DKFZ, Heidelberg, Germany) in the129/Sv genetic background. The mice were backcrossed for 4 generationsonto the Balb/c(H-2^(d)) background in order to take advantage of theknock-in transgene in a murine background where tumors and otherreagents are readily available. The hupki mice on the Balb/c backgroundwere backcrossed to homozygosity as confirmed by PCR analysis, using amating procedure that minimized inbreeding effects (data not shown). The4T1 (H-2^(d)) murine breast carcinoma cell line was stably transfectedwith human p53, and hupki mice were s.c. injected with 5×10⁴ 4T1/hup53in the flank. Mice injected with 4T1/hup53 grow progressive tumors, andthe majority succumb to these tumors by day 35. To test the efficacy ofrMVAhup53, mice were vaccinated with 10⁷ pfu rMVAhup53 by i.p. injectionon day 6 after 4T1/hup53 injection. Ten days later, the mice received anrMVAhup53 booster injection, along with CpG-ODN (15 nmole of ODN 1826)and anti-CTLA-4 mAb (50 μg/mouse). rMVAhup53 vaccination resulted in astatistically significant improvement in survival (p<0.05, two sidedT-test) compared to PBS controls (FIG. 19).

As stated above, the foregoing are merely intended to illustrate thevarious embodiments of the present invention. As such, the specificmodifications discussed above are not to be construed as limitations onthe scope of the invention. It will be apparent to one skilled in theart that various equivalents, changes, and modifications may be madewithout departing from the scope of the invention, and it is understoodthat such equivalent embodiments are to be included herein. Allreferences cited herein are incorporated by reference as if fully setforth herein.

Abbreviations used herein: GFP, green fluorescent protein; DC, dendriticcells; IFN-γ^(KO), IFN-γ knock out; MVA, modified vaccinia virus Ankara;rMVA, recombinant modified vaccinia virus Ankara; rAd-mup53, recombinantAdenovirus expressing murine wild type p53; hup53, wild type human p53;mup53, wild type murine p53; rMVAp53, recombinant MVA expressing p53;rMVAmup53, recombinant MVA expressing wild type murine p53; rMVAhup53,recombinant MVA expressing wild type human p53; rMVApp65, recombinantMVA expressing pp65; rVVmup53, recombinant vaccinia virus expressingmurine wild type p53; rWpp65, recombinant vaccinia virus expressingpp65; wtMVA, wild type MVA; WR, Western Reserve; i.p., intraperitoneal;s.c., subcutaneous; mAb, monoclonal antibody.

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1. A composition comprising a recombinant MVA virus (rMVA) containing anucleic acid sequence encoding wild-type human p53, wherein said nucleicacid sequence comprises the nucleotide sequence of SEQ ID NO:2.
 2. A kitcomprising the composition of claim 1 and instructions foradministration of said composition to a subject having a p53-expressingmalignancy.
 3. The kit of claim 2, wherein said subject is human.
 4. Acomposition comprising a recombinant MVA virus (rMVA) containing anucleic acid sequence encoding wild-type human p53 wherein saidwild-type human p53 is encoded by the nucleotide sequence of SEQ IDNO:2. and wherein said wild-type human p53 is capable of arresting thecell cycle.
 5. A kit comprising the composition of claim 4 andinstructions for administration of said composition to a subject havinga p53-expressing malignancy.